A central tenet of eukaryotic cell biology is that DNA replication must be tightly controlled so that it occurs only once per cell cycle. It is presumed, but largely untested, that this control is vital for preserving genome integrity. Our long-term goal is to understand how the re-initiation of DNA replication is reliably prevented at the thousands of replication origins scattered throughout eukaryotic genomes, and to discern the effect of disrupting this control on genome stability. We study replication control in budding yeast because this model system offers an exceptional opportunity to dissect the complex, overlapping mechanisms that are required to achieve this control with such extraordinary fidelity. Additionally, the molecular genetic tools available in budding yeast allow us to apply both simple and sophisticated technologies to query the effects of disrupting replication controls. In previous funding periods, we demonstrated that cyclin-dependent kinases (CDKs) use multiple overlapping mechanisms to prevent origins from re-initiating within a single cell cycle. We have also shown that re-replication arising from loss of these controls leads to significant chromosomal breakage and lethality, providing a previously unknown justification for the importance of replication control. More recently, we provided the first evidence that re-replication is a highly efficient means to induce gene amplification (Green et al, Science, in press). Re-replication induced gene amplification (RRIGA) occurred with extraordinary efficiency (roughly 1/20 re-initiation events). This finding supports the compelling hypothesis that even minor impairment of replication control may contribute to genome instability. Ultimately, we hope to demonstrate that re-replication can drive the copy number changes observed in tumorigenesis, human genetic variation, and evolution. Here we propose to expand our understanding of how the loss of replication control leads to genomic instability, both by probing the mechanisms that underlie RRIGA as well as by further investigating the biological consequences and significance of loss of replication control. We propose to (1) define the mechanism and parameters enabling RRIGA;(2) determine how local regulatory factors modulate replication control at origins that are highly susceptible to re-initiation;(3) determine whether RRIGA participates in a model of evolutionary adaptation;and (4) establish whether re-replication can induce chromosome missegregation. These data will significantly enhance our understanding of how re-replication promotes genomic instability, as well as give insight into the biological significance of loss of replication control. PUBLIC HEALTH RELEVANCE: Abnormal genetic changes underlie the formation of cancer cells and can lead to human genetic diseases, but the sources of these changes is poorly understood. Our research in a yeast model organism is establishing important connections between these diseases and the control of DNA replication, which normally ensures that every segment of DNA is replicated exactly once each time a cell divides. Our proposed project will investigate why inappropriate re-replication arising from the loss of replication control is so amazingly efficient at inducing the types of genetic changes observed in cancer and genetic disorders. These findings should provide strong impetus for cancer biologists and human geneticists to investigate the role of replication dysregulation in their fields.